This invention relates to the field of nucleic acid amplification and detection. More particularly, the invention provides methods, compositions and kits for amplifying (i.e., making multiple copies of) nucleic acid sequences and for detecting amplified sequences.
A number of target amplification methods have been developed which permit the implementation of sensitive diagnostic assays based on nucleic acid detection. They include the polymerase chain reaction (PCR) (U.S. Pat. Nos. 4,683,195 and 4,683,202), ligase chain reaction (LCR) (See, for example, Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991)); and U.S. Pat. No. 5,494,810), self-sustained sequence replication (3SR) and nucleic acid sequence based amplification (NASBA) (See, for example, Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878 (1990)). Loop-mediated isothermal amplification (LAMP) (WO 00/28082), strand displacement amplification (SDA) (See, for example, U.S. Pat. Nos. 5,455,166 and 5,470,723), Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) method described in International Publication WO02/16639, Rolling Circle Amplification (RCA) (U.S. Pat. Nos. 5,714,320 and 6,235,502), and the like.
The real-time polymerase chain reaction (PCR) is carried out in a closed-tube format and can be used to obtain quantitative results. Several methods using a labeled sequence-specific probe are currently available for performing real-time PCR, such as TaqMan® probes (U.S. Pat. Nos. 5,210,015 and 5,487,972, and Lee et al., Nucleic Acids Res. 21:3761-6, 1993); molecular beacons (U.S. Pat. Nos. 5,925,517 and 6,103,476, and Tyagi and Kramer, Nat. Biotechnol. 14:303-8, 1996); self-probing amplicons (scorpions) (U.S. Pat. No. 6,326,145, and Whitcombe et al., Nat. Biotechnol. 17:804-7, 1999); Amplisensor (Chen et al., Appl. Environ. Microbiol. 64:4210-6, 1998); Amplifluor (U.S. Pat. No. 6,117,635, and Nazarenko et al., Nucleic Acids Res. 25:2516-21, 1997 and U.S. Pat. No. 6,117,635); displacement hybridization probes (Li et al., Nucleic Acids Res. 30:E5, 2002); DzyNA-PCR (Todd et al., Clin. Chem. 46:625-30, 2000); fluorescent restriction enzyme detection (Cairns et al. Biochem. Biophys. Res. Commun. 318:684-90, 2004); and adjacent hybridization probes (U.S. Pat. No. 6,174,670 and Wittwer et al., Biotechniques 22:130-1, 134-8, 1997).
Strand displacement reaction has been implemented in a number of amplification methods. These methods include SDA, LAMP, ICAN, RCA, and the like. RCA has been further developed in a technique, named Multiple Displacement Amplification (MDA), which generates a whole genome amplification (See, for example, U.S. Pat. No. 6,124,120 and Dean et. al., Proc. Natl. Acad. Sci. USA 99:5261-5266 (2002)). Another method named isothermal strand displacement nucleic acid amplification has been developed (see U.S. Pat. Nos. RE38,960E, RE39,007E). These methods use strand displacement, multiple primers and isothermal conditions to achieve a substantial amplification. However, these methods suffer from a number of drawbacks such as difficulty of quantification, slow reaction, non-specific spurious product, low sensitivity etc.
PCR remains the most widely used DNA amplification and quantitation method. Nested PCR, a two-stage PCR, is used to increase the specificity and sensitivity of the PCR (U.S. Pat. No. 4,683,195). Nested primers for use in the PCR amplification are oligonucleotides having sequence complementary to a region on a target sequence between reverse and forward primer targeting sites. However, PCR in general has several limitations. PCR amplification can only achieve less than two fold increase of the amount of target sequence at each cycle. It is still relatively slow. The sensitivity of this method is typically limited, making it difficult to detect target that may be present at only a few molecules in a single reaction.
Current technologies for generating single stranded amplification end product include PCR based methods such as asymmetric PCR (Gyllensten et al., Proc. Natl. Acad. Sci. USA 85:7652-7656, 1988), LATE-PCR (Sanchez et al., Proc. Natl. Acad. Sci. USA 101:1933-1938, 2004), and a method described in BioTechniques, 40:759-763, 2006 by Tang et al. A non-PCR based method called polynomial amplification of nucleic acids has been developed (U.S. Pat. No. 7,112,406). Due to the non-exponential nature of amplification, the polynomial amplification could reduce levels of carry-over contamination. However this is at the expense of amplification efficiency. Overrall, all these methods are inefficient and the generated single stranded amplification end product is not suitable for monitoring the amplification process and is not suitable for real-time detecting and quantification of amount of target present in a sample.
Thus it can be seen that novel methods and materials addressing one or more of these drawbacks would provide a contribution to the art.